Methods and compositions for modulating transport of a drug

ABSTRACT

Methods and pharmaceutical compositions for inhibiting or decreasing transport of a drug by a transporter of multidrug resistance-associated protein comprising a compound of Formula (VI) 
                         
wherein R 1  and R 2  are small peptides or modified peptides, are provided. The methods and compositions are useful in enhancing efficacy of drugs such as anti-inflammatory agents, neurological agents, thyroid agents, ocular agents, cancer chemotherapeutics, antibiotics, antimicrobials, antivirals and protease inhibitors to treat human immunodeficiency virus.

This invention was made with government support under grant numbersAR055073, DK080774 and DK093903 awarded by the National Institutes ofHealth. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for modulatingtransport of a drug by a transporter of multidrug resistance-associatedprotein via administration of an agent which crosslinks glutathione in acell or a compound comprising crosslinked small peptides or modifiedpeptides.

BACKGROUND

Efflux transporters are proteins that span the plasma membrane of a celland catalyze the export of compounds from these cells at the expense ofATP. The first ABC transporter described was multidrug resistanceprotein 1 (MDR1 in human, Mdr1a/b in rodents, or P-glycoprotein; Julianoet al. Biochimica et biophysica acta 1976 455(1):152-162). This 170kilodalton transmembrane protein, encoded by the gene name ABCB1, spansthe plasma membrane six times in two distinct regions (Van der Bliek etal. Cancer Res 1988 48(21):5927-5932). When this protein was firstcharacterized, it was discovered that cells overexpressing Mdr1a/b wereresistant to colchicine, doxorubicin, and actinomycin D (Devault, A &Gros, P Molecular and cellular biology 1990 10(4):1652-1663). Followingthe discovery of MDR1/Mdr1, it was realized that efflux pumps exist thatdesensitize cells to cancer chemotherapy; however, there are manycancers that do not overexpress this ATPase (Klaassen et al. PharmacolRev 2010 62(1):1-96). Because of this resistance of many tumors tochemotherapy, the discovery of additional xenobiotic efflux pumps waspursued, leading to the discovery and characterization of multidrugresistance-associated protein 1 (MRP1/Mrp1; Cole et al. Science 1992258(5088):1650-1654). Soon thereafter, MRP1 was linked to resistance toanticancer drugs (Stride et al. Mol Pharmacol 1997 52(3):344-353; Grantet al. Cancer Res 1994 54(2):357-361; and Barrand et al. Journal of theNational Cancer Institute 1994 86(2):110-117).

MRP1/Mrp1 is known to transport glutathione conjugates of nitrogenmustard-derived compounds chlorambucil and melphalan (Barnouin et al.British journal of cancer 1998 77(2):201-209; Jedlitschky et al. Cancerresearch 1996 56(5):988-994; and Paumi et al. The Journal of biologicalchemistry 2001 276(11):7952-7956). Other ligands for MRP1/Mrp1 includeoxidized glutathione (Minich et al. Journal of neurochemistry 200697(2):373-384), the topoisomerase II inhibitors doxorubicin, idarubicin(Smeets et al. Leukemia 1999 13(9):1390-1398), and etoposide (Lorico etal. Cancer research 1995 55(19):4352-4360; Tasaki et al. The Journal ofurology 1995 154(3):1210-1216; Brock et al. Cancer research 199555(3):459-462; Abe et al. International journal of cancer Journalinternational du cancer 1994 58(6):860-864; Kubota et al. Cancerchemotherapy and pharmacology 1994 34(3):183-190; Schneider et al.Cancer research 1994 54(1):152-158; Hamaguchi et al. Cancer research1993 53(21):5225-5232; Godinot et al. Molecular cancer therapeutics 20032(3):307-316), the lipid peroxidation product glutathione-conjuguated4-hydroxynonenal (Renes et al. The Biochemical journal 2000 350 Pt2:555-561; Sultana R, & Butterfield D A Neurochemical research 200429(12):2215-2220), glutathione-conjugated aflatoxin B1,estradiol-17β-glucoronide (Qian et al. The Journal of biologicalchemistry 2001 276(9):6404-6411), and glutathione-conjugatedmethylmercury (Klaassen et al. Pharmacol Rev 2010 62(1):1-96; Rush etal. Neurotoxicology 2012 33(3):476-481; and Suzuki et al. Seminars inliver disease 1998 18(4):359-376). Some heavy metals have also beenshown to act as substrates for human MRP1, including antimony salts(Gayet et al. FEBS letters 2006 580(30):6891-6897; Mookerjee et al.Antimicrobial agents and chemotherapy 2008 52(3):1080-1093), themercuric ion (Aleo et al. Toxicology 2005 206(1):137-151), arsenate, andarsenite (Vernhet et al. Toxicology 2000 142(2):127-134; Leslie et al.The Journal of biological chemistry 2004 279(31):32700-32708). Even forcompounds transported by MRP1/Mrp1 that are not conjugated toglutathione, co-transport of glutathione, or in some cases, S-methylglutathione (Rothnie et al. The Journal of biological chemistry 2006,281(20):13906-13914), is still required for MRP1/Mrp1 to function(Leslie et al. The Journal of biological chemistry 2001,276(30):27846-27854). Nine human MRPs have been characterized; anadditional MRP transporter, MRP2/Mrp2 is also known to mediateextracellular transport of glutathione-conjugated electrophiles(Klaassen C D & Aleksunes L M Pharmacol Rev 2010, 62(1):1-96). MRP2,like MRP1, has been reported to transport glutathione-conjugatedchlorambucil, although less efficiently than MRP1 (Smitherman et al. TheJournal of pharmacology and experimental therapeutics 2004,308(1):260-267); it is also known to efflux cisplatin, a crosslinkingagent with a generally similar molecular mechanism of action as thenitrogen mustard HN2 when cisplatin is conjugated to glutathione (Wen etal. Am J Pathol 2014, 184(5):1299-1308).

The human MRP1 gene was first isolated from a doxorubicin-resistantsmall-cell lung carcinoma, and it was determined to be a member of theATP-binding cassette family based on its primary sequence (Cole et al.Science 1992, 258(5088):1650-1654). This transmembrane protein isoverexpressed in multidrug resistant cervical cancer HeLa cells andnon-small cell lung carcinoma cell lines (Cole et al. Science 1992,258(5088):1650-1654). It has also been demonstrated that MRP1 wasoverexpressed in a number of human tumor cell lines that do not expressMDR1 but still possess the “multidrug resistant” phenotype. Two gliomacell lines, IN500 and T98G, have elevated MRP1 expression and areresistant to etoposide, vincristine, and doxorubicin and have adecreased accumulation of etoposide following treatment (Abe et al.International journal of cancer Journal international du cancer 1994,58(6):860-864; Benyahia et al. Journal of neuro-oncology 2004,66(1-2):65-70). Various inhibitors of MRP1 activity were shown toreverse resistance to etoposide and doxorubicin in human glioma cells(Abe et al. British journal of cancer 1995, 72(2):418-423). Clinically,high-grade gliomas have been demonstrated to express more MRP1 thanthose of a lower grade (de Faria G P et al. Cancer investigation 2008,26(9):883-889). Research has shown a correlation between MRP1 expressionand a poor prognosis for patients with breast cancer (Filipits et al.Anticancer research 1999, 19(6B):5043-5049; Abaan et al. Cancerinvestigation 2009, 27(2):201-205), ovarian cancer (Faggad et al.Histopathology 2009, 54(6):657-666), and neuroblastoma [52-55].Neuroblastoma cells were also shown to have an increased MRP1 expressioncompared to healthy cells (Peaston et al. British journal of cancer2001, 85(10):1564-1571). A correlation was also shown between MRP1expression and a poor response to treatment chemotherapy in acutelymphocytic leukemia, though this study combined several differenttreatment regimens, some of which included MRP1/Mrp1 substrates and someof which did not (Styczynski et al. Journal of cancer research andclinical oncology 2007, 133(11):875-893). A further study of acutelymphocytic lymphoma examined patients treated with the BFM-95 protocolthat includes MRP1/Mrp1 ligands methotrexate, vincristine, daunorubicin,and cyclophosphamide, and a statistically significant correlation wasfound between MRP1 expression and poor response to therapy (Kourti etal. International journal of hematology 2007, 86(2):166-173). Clinicalresearch also showed that MRP1, as well as MDR1, MRP2, and MRP3 areelevated in residual tumors following treatment with MRP1 substratedoxorubicin compared with the untreated primary tumors (Tada et al.International journal of cancer Journal international du cancer 2002,98(4):630-635).

It has been suggested, as well as observed, that multiple drugs are moreeffective than just one compound when treating tumors. In fact,treatment with etoposide and cisplatin or chlorambucil has been thestandard of care to treat B cell lymphoma for many years (Keating G M,BioDrugs—: clinical immunotherapeutics, biopharmaceuticals and genetherapy 2011, 25(1):55-61). The combination of cyclophosphamide,doxorubicin, vincristine, and prednisone, also known as CHOP, iscommonly used and effective in combating non-Hodgkin's lymphoma (Cullenet al. The New England journal of medicine 2005, 353(10):988-998).

Cyclophosphamide is a nitrogen mustard-related compound that has,similar to chlorambucil and melphalan, been hypothesized to be apotential substrate for MRP1/Mrp1 once conjugated to glutathione (Zhanget al. Drug metabolism reviews 2005, 37(4):611-703). In fact, acorrelation was observed between MRP1 expression and poor response tocyclophosphamide for breast cancer patients using this drug (Filipits etal. Journal of clinical oncology: official journal of the AmericanSociety of Clinical Oncology 2005, 23(6):1161-1168). Vincristine anddoxorubicin, two other compounds that are included in CHOP, are alsoknown substrates for MRP1/Mrp1 (Abe et al. British journal of cancer1995, 72(2):418-423; van Tellingen et al. British journal of cancer2003, 89(9):1776-1782; de Cremoux et al. Pediatric blood & cancer 2007,48(3):311-317; and Kang et al. Anticancer research 1997,17(5A):3531-3536). Despite the fact that it has been known for some timethat treatment with multiple cancer drugs is more efficacious thantreatment with just one drug, and the impact can often be greater thanan additive effect, the mechanism for such an interaction betweentreatments has remained elusive (Rubin P: Jama 1973, 223(2):164-166;Tomashefsky et al. Oncology 1964, 17:1-6; and Nicholson et al. Britishmedical journal 1970, 3(5713):7-10). It has long been thought that thenitrogen mustard HN2 can sensitize tumors to other cancerchemotherapeutic agents simply by causing DNA damage (Cheson B D & LeoniL: Clinical advances in hematology & oncology: H&O 2011, 9(8 Suppl19):1-11).

Glutathione-conjugated electrophiles are common substrates forMRP1/Mrp1. Glutathione-conjugated ethacrynic acid derivatives have beendisclosed to inhibit MRP1 in the low micromolar range (Burg et al.Molecular pharmacology 2002, 62(5):1160-1166). Triazole-bridged dimershave also been disclosed to be effective inhibitors of MRP1 in thenanomolar (73 to 133) range (Wong et al. J Med Chem 2009,52(17):5311-5322).

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a method for inhibiting ordecreasing transport of a drug by a transporter of multidrugresistance-associated protein. In this method, an agent which crosslinksglutathione in a cell or a compound comprising crosslinked smallpeptides or modified peptides is administered to the cell therebyinhibiting or decreasing transport of a drug by a transporter ofmultidrug resistance-associated protein in the cell. In one nonlimitingembodiment, the method further comprises administering a drug prior to,in combination or following administration of the agent or compound. Inone nonlimiting embodiment, efficacy of the drug is dependent onconcentrations inside the cell. In one nonlimiting embodiment, the drugis a substrate for the transporter of multidrug resistance-associatedprotein.

Another aspect of the present invention relates to a method forpotentiating the action of a drug on a cell. In this method, an agentwhich crosslinks glutathione in a cell or a compound comprisingcrosslinked small peptides or modified peptides is administered to thecell thereby inhibiting or decreasing transport of a drug by atransporter of multidrug resistance-associated protein in the cell. Theagent or compound is administered prior to, in combination with, orfollowing administration of a drug. In one nonlimiting embodiment,efficacy of the drug is dependent on concentrations inside the cell. Inone nonlimiting embodiment, the drug is a substrate for the transporterof multidrug resistance-associated protein.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition for inhibiting or decreasing transport of a drug by atransporter of multidrug resistance-associated protein in a cell. Thecomposition comprises an agent which crosslinks glutathione in the cellor a compound comprising crosslinked small peptides or modified peptidesand a pharmaceutically acceptable carrier. In one nonlimitingembodiment, the composition further comprises a drug that is a substratefor the transporter of multidrug resistance-associated protein. In onenonlimiting embodiment, the composition further comprises a drug,efficacy of which is dependent on concentrations inside the cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A through 1C are linegraphs showing inhibition of MRP1 activity inA549 tumor cells following exposure to divinyl sulfone (FIG. 1A),diethyl acetylenedicarboxylate (FIG. 1B) and the nitrogen mustardreferred to herein as HN2 (FIG. 1C). In these experiments, A549 cellswere treated with increasing concentrations of divinyl sulfone, diethylacetylenedicarboxylate or HN2. Each chemical reacts in cells withglutathione (GSH) to form a bifunctional derivative. Calcein, afluorescent MRP1 substrate, was used to measure the effects ofinhibitors on MRP1 activity. Each compound inhibited MRP1 activity inA549 tumor cells in the nanomolar concentration range.

FIG. 2 is a bargraph showing the effects of HN2 on the MRP1 transporter.Bimane-GS, a fluorescent MRP1 substrate, was incorporated into insideout membrane vesicles isolated from insect cells overexpressing the MRP1transporter. Treatment with 100 nM HN2 inhibited transporter activity.Reaction mixture containing vesicles were supplemented with 5 mM GSH.

FIG. 3A through 3C are linegraphs showing the effects of HN2 on growthinhibition of A549 tumor cells by cancer chemotherapeutic agents thatare substrates for MRP1. Cells were treated with non-cytotoxicconcentrations of HN2 (10 nM) and increasing concentrations of etoposide(FIG. 3A), methotrexate (FIG. 3B) or vincristine (FIG. 3C). HN2, byreacting with GSH and forming a bifunctional adduct, inhibited drugefflux and markedly enhanced sensitivity to the chemotherapeutic agents.

FIG. 4A and FIG. 4B are linegraphs showing the effects of etoposide onHN2-induced growth inhibition in HEK cells overexpressing MRP1. Cellswere treated with non-cytotoxic concentrations of HN2 (10 nM), followedby increasing concentrations of etoposide. Initial treatment with HN2blocked the MRP1 efflux transporter in HEK cells overexpressing MRP1(HEK MRP1) and increased their sensitivity to further treatments withHN2. FIG. 4A shows HEK cells overexpressing MRP1. FIG. 4B shows controlcells with little or no MRP1.

DETAILED DESCRIPTION OF THE INVENTION

A large number of substrates for MRP1/Mrp1 are drugs wherein clinicalefficacy is limited by MRP1-mediated efflux transport. Accordingly,pharmacological inhibition of MRP1 and other transporters of multidrugresistance-associated protein could serve as a means of increasing thesensitivity as well as specificity of such drugs.

It has now been found that agents which crosslink glutathione in a cellor compounds comprising crosslinked small peptides or modified peptidescan be administered to a cell thereby inhibiting or decreasing transportof a drug by a transporter of multidrug resistance-associated protein inthe cell. Accordingly, the present invention relates to compositions andmethods for inhibiting or decreasing transport of a drug by atransporter of multidrug resistance-associated protein and/orpotentiating the action of a drug on a cell.

By “cell” as used herein, it is meant to encompass cells in vitro, exvivo as well as in vivo. In one nonlimiting embodiment, the cell is in atumor in a human or other animal or is a microbe infecting a human orother animal.

By “transporter of multidrug resistance-associated protein” as usedherein, it is meant to be inclusive of, but is not limited to, MRP1,MRP2, MRP3, MRP4 and MRP6.

Agents useful in the present invention must be capable of crosslinkingglutathione and must be of a size and structure which does not interferewith the ability of the agent, when crosslinked with glutathione, toinhibit a transporter of multidrug resistance-associated protein.Examples of crosslinking agents useful in the present invention include,but are in no way limited to, divinyl sulfone, diethylacetylenedicarboxylate, nitrogen mustards such as, but not limited tomechlorethamine and HN2, cisplatin and muconaldehyde. Additionalcrosslinking agents useful and within the scope of the present inventioncan be routinely selected by the skilled artisan based upon teachingsherein. Nonlimiting examples of additional commercially availablecrosslinking agents which can be used in the present invention aredisclosed in Thermo Scientific's Crosslinking Technical Handbook.

Compounds useful in the present invention comprise crosslinked smallpeptides or modified peptides.

By “small peptides”, as used herein, it is meant a peptide of less than20 amino acids, more preferably less than 10 amino acids and morepreferably about 1 to 5 amino acid residues in length. In onenonlimiting embodiment, one or both of the small peptides is glutathioneor a peptide similar in size and structure to glutathione with anucleophilic thiol group on one end for conjugation to electrophiles. Anonlimiting example of a peptide similar in size and structure toglutathione which can be used is N-acetyl cysteine.

Nonlimiting examples of compounds useful in the present inventioninclude

wherein R₁ and R₂ are small peptides or modified peptides. As will beunderstood by the skilled artisan upon reading this disclosure,compounds comprising alternative crosslinking agents to those of formula(I), (II) or (III), with a size and structure which does not interferewith the ability of the compound to inhibit a transporter of multidrugresistance-associated protein, can be used and are within the scope ofthe present invention. Nonlimiting examples of additional commerciallyavailable crosslinking agents which could be useful in the compounds ofthe present invention are disclosed in Thermo Scientific's CrosslinkingTechnical Handbook.

In one nonlimiting embodiment, the agent or compound is administeredeffectively at a low concentration to inhibit or decrease transport of adrug by a transporter of multidrug resistance-associated protein and/orpotentiate the action of a drug on a cell. By “low concentration” asused herein, it is meant a non-cytotoxic concentration.

In one nonlimiting embodiment, the agent or compound is co-administeredwith a drug. By “co-administered” as used herein, it is meantadministration of the agent or compound prior to, simultaneously orsubsequent to administration of the drug. In one nonlimiting embodiment,efficacy of the administered drug is dependent upon its concentrationsinside a cell. In one nonlimiting embodiment, inhibiting or decreasingtransport of the drug by the transporter of multidrugresistance-associated protein via coadministration of the agent orcompound potentiates action of the drug on the cell. In one nonlimitingembodiment, the drug is a substrate for a transporter of multidrugresistance-associated protein. Examples of drugs which can beadministered with the agents or compounds in accordance with the presentinvention include, but are not limited to, anti-inflammatory agents,neurological agents, thyroid agents, ocular agents, cancerchemotherapeutics, antibiotics, antimicrobials, antivirals and proteaseinhibitors to treat human immunodeficiency virus.

Cells to which the agents and compounds of the present invention can beadministered to include, but are in no way limited to, cancer cells andmicrobes.

Also provided by the present invention are pharmaceutical compositionsfor inhibiting or decreasing transport of a drug by a transporter ofmultidrug resistance-associated protein in a cell. The pharmaceuticalcompositions of the present invention comprise an agent which crosslinksglutathione in the cell as described herein and/or a compound comprisingcrosslinked small peptides or modified peptides and a pharmaceuticallyacceptable carrier as described herein. In one nonlimiting embodiment,the pharmaceutical composition further comprises a drug. In onenonlimiting embodiment, the drug is a substrate for a transporter ofmultidrug resistance-associated protein. In one nonlimiting embodiment,efficacy of the drug is dependent on concentrations inside the cell.Nonlimiting examples of drugs which can be included in the presentinvention include anti-inflammatory agents, neurological agents, thyroidagents, ocular agents, cancer chemotherapeutics, antibiotics,antimicrobials, antivirals and protease inhibitors to treat humanimmunodeficiency virus.

Pharmaceutical compositions of the present invention can be administeredby various routes depending upon the condition or disease to be treated.It is expected that these compositions will be effective followingsystemic as well as local administration. Accordingly, thepharmaceutical compositions of the invention may be administeredsystemically or locally, and by any suitable route such as oral, buccal,sublingual, transdermal, inhalation, subcutaneous, intraocular,intravenous, intramuscular, intrathecally, epidurally or intraperitonealadministration, and the like (e.g., by injection). Preferably, the agentwhich crosslinks glutathione in the cell as described herein and/or acompound comprising crosslinked small peptides or modified peptides andthe drug are administered simultaneously via the same route ofadministration. However, it is expected that administration of the agentwhich crosslinks glutathione in the cell as described herein and/or thecompound comprising crosslinked small peptides or modified peptides andthe drug separately, via the same route or different route ofadministration, within a time frame during which each remains active,will also be effective therapeutically as well as in inhibiting ordecreasing transport of the drug by a transporter of multidrugresistance-associated protein in cells. Further, it is expected thatadministration of the agent which crosslinks glutathione in the cell asdescribed herein and/or the compound comprising crosslinked smallpeptides or modified peptides already receiving the drug will inhibit ordecrease transport of the drug by a transporter of multidrugresistance-associated protein in cells. Thus, treatment with the agentwhich crosslinks glutathione in the cell as described herein and/or thecompound comprising crosslinked small peptides or modified peptides andthe drug need not begin at the same time. For example, administration ofthe agent which crosslinks glutathione in the cell as described hereinand/or the compound comprising crosslinked small peptides or modifiedpeptides may begin several days, weeks, months or more before or aftertreatment with the drug.

Accordingly, for purposes of the present invention, the agent whichcrosslinks glutathione in the cell as described herein and/or thecompound comprising crosslinked small peptides or modified peptides andthe drug, can be administered together in a single pharmaceuticallyacceptable vehicle or separately, each in their own pharmaceuticallyacceptable vehicle.

As used herein “pharmaceutically acceptable vehicle” includes any andall solvents, excipients, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, as well as liposomes, which are compatible with the activity ofthe agent which crosslinks glutathione in the cell as described hereinand/or the compound comprising crosslinked small peptides or modifiedpeptides and the drug and are physiologically acceptable to a subject.An example of a pharmaceutically acceptable vehicle is buffered normalsaline (0.15 M NaCl). Liposomes include water-in-oil-in-water CGFemulsions such as described in U.S. Pat. No. 5,891,468 as well asconventional liposomes. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theagent which crosslinks glutathione in the cell as described hereinand/or the compound comprising crosslinked small peptides or modifiedpeptides or drugs, use thereof in the compositions suitable forpharmaceutical administration is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

Carrier or substituent moieties useful in the present invention may alsoinclude moieties which allow the agent which crosslinks glutathione inthe cell as described herein and/or the compound comprising crosslinkedsmall peptides or modified peptides and the drug to be selectivelydelivered to a target organ or cell. Many targeting moieties are known,and include, but are in no way limited to, asialoglycoproteins (seee.g., Wu, U.S. Pat. No. 5,166,320) and other ligands which aretransported into cells via receptor-mediated endocytosis.

Solid dosage forms for oral administration include ingestible capsules,tablets, pills, lollipops, powders, granules, elixirs, suspensions,syrups, wafers, buccal tablets, troches, and the like. In such soliddosage forms the agent which crosslinks glutathione in the cell asdescribed herein and/or the compound comprising crosslinked smallpeptides or modified peptides and/or the drug is mixed with at least oneinert, pharmaceutically acceptable excipient or diluent or assimilableedible carrier such as, but not limited to, sodium citrate or dicalciumphosphate and/or fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, humectants such as glycerol,disintegrating agents such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate,solution retarding agents such as paraffin, absorption accelerators suchas quaternary ammonium compounds, wetting agents such as, for example,cetyl alcohol and glycerol monostearate, absorbents such as kaolin andbentonite clay, and/or lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and any mixtures thereof, or incorporated directly into a subject'sdiet. In the case, of capsules, tablets and pills, the dosage form mayalso comprise buffering agents. Solid compositions of a similar type mayalso be employed as fillers in soft and hard-filled gelatin capsulesusing such excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like. The percentage of the agentwhich crosslinks glutathione in the cell as described herein and/or thecompound comprising crosslinked small peptides or modified peptidesand/or the drug in the compositions and preparations may, of course, bevaried. The amount of the agent which crosslinks glutathione in the cellas described herein and/or the compound comprising crosslinked smallpeptides or modified peptides and/or the drug in such therapeuticallyuseful compositions is such that a suitable dosage will be obtained.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well-known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. The agent which crosslinksglutathione in the cell as described herein and/or the compoundcomprising crosslinked small peptides or modified peptides and/or thedrug can also be in micro-encapsulated form, if appropriate, with one ormore of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the agent which crosslinks glutathione in the cell asdescribed herein and/or the compound comprising crosslinked smallpeptides or modified peptides and/or the drug, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oils (in particular, cottonseed, ground nutcorn, germ olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the agent which crosslinks glutathione inthe cell as described herein and/or the compound comprising crosslinkedsmall peptides or modified peptides and/or the drug, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

The pharmaceutical compositions of the present invention can beadministered topically. For topical use the agent which crosslinksglutathione in the cell as described herein and/or the compoundcomprising crosslinked small peptides or modified peptides and/or thedrug can be prepared in suitable forms to be applied to the skin, ormucus membranes of the nose and throat, and can take the form oflotions, creams, ointments, liquid sprays or inhalants, drops,tinctures, lozenges, or throat paints. Such topical formulations furthercan include chemical compounds such as dimethylsulfoxide (DMSO) tofacilitate surface penetration of the active ingredient. In othertransdermal formulations, typically in patch-delivered formulations, thepharmaceutically composition is formulated with one or more skinpenetrants, such as 2-N-methyl-pyrrolidone (NMP) or Azone. Forapplication to the eyes or ears, the compounds of the present inventioncan be presented in liquid or semi-liquid form formulated in hydrophobicor hydrophilic bases as ointments, creams, lotions, paints or powders.

For rectal administration, the agent which crosslinks glutathione in thecell as described herein and/or the compound comprising crosslinkedsmall peptides or modified peptides and/or the drug can be administeredin the form of suppositories admixed with conventional carriers such ascocoa butter, wax or other glyceride.

Inhalation formulations can also readily be formulated. For inhalation,various powder and liquid formulations can be prepared. For aerosolpreparations, a sterile formulation of the agent which crosslinksglutathione in the cell as described herein and/or the compoundcomprising crosslinked small peptides or modified peptides and/or thedrug may be used in inhalers, such as metered dose inhalers, andnebulizers. Aerosolized forms may be especially useful for treatingrespiratory disorders.

Pharmaceutical compositions according to the invention are administeredat a therapeutically effective dosage sufficient to achieve the desiredtherapeutic effect of inhibiting or decreasing transport of the drug bya transporter of multidrug resistance-associated protein in cells.Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject, compositions, and mode ofadministration. The selected dosage level will depend upon the activityof the particular compound, the route of administration, the severity ofthe condition being treated, the condition and prior medical history ofthe subject being treated, the age, sex, and weight of the subject, andthe ability of the therapeutic compound to produce the desiredtherapeutic effect in the subject. Dosage regimens can be adjusted toprovide the optimum therapeutic response.

The ability of various compounds of the present invention to inhibit MRPactivity was demonstrated. In these experiments, the compounds divinylsulfone, diethyl acetylenedicarboxylate, and the nitrogen mustard HN2were demonstrated to inhibit MRP activity in A549 cells. See FIG. 1A,FIG. 1B and FIG. 1C. Treatment of A549 cells with divinyl sulfoneinhibited the efflux of MRP1 substrate calcein (see FIG. 1A) with anIC₅₀ of 5.8 nM. Similar effects were seen following exposure to diethylacetylenedicarboxylate (see FIG. 1B; IC₅₀=6.0 nM) and HN2 (see FIG. 10,IC₅₀=5.5 nM).

HN2 was also demonstrated to inhibit MRP1 activity in HEK cellsoverexpressing MRP1. More specifically, significant inhibition of MRP1functional activity was observed after treatment with 2 nM and 4 nM HN2.The efflux of calcein in the HEK control cells was significantly lowerthan that of HN2, and this background efflux was not inhibited by HN2.HN2 also inhibited efflux of calcein in HEK MRP2 cells, but 30 nM HN2was required for significant inhibition of MRP2 function.

HN2 also inhibited uptake of fluorescent MRP1 substrate bimane-GS ininverted membrane vesicles prepared from Sf9 cells overexpressing humanMRP1 in the nanomolar range where the assay was supplemented with 5 mMreduced glutathione to allow formation of bifunction cross-linked HN2(see FIG. 2).

Sub-cytotoxic concentrations of HN2 were also demonstrated to sensitizetumors to another anticancer drug that can act as a substrate for MRP1.Such efficacy should prove useful in limiting the concentration used forvarious cancer chemotherapeutic compounds to thereby limit side effects.Specifically, it was investigated whether a sub-cytotoxic concentrationof HN2 could be employed to sensitize cells to etoposide, methotrexate,or vincristine. Etoposide is an anticancer agent that causescytotoxicity through inhibition of topoisomerase II, leading to DNAstrand breaks that has long been thought to be exported from tumor cellsby MRP1/Mrp1. Methotrexate is an inhibitor of dihydrofolate reductasethat also is implicated as a substrate for MRP1/Mrp1. Vincristine is amicrotubule-disrupting anti-mitotic compound also transported byMRP1/Mrp1. Etoposide, methotrexate or vincristine were found to inhibitgrowth of A549 cells (FIGS. 3A, 3B and 3C, respectively). However,co-treatment of the cells with a sub-cytotoxic concentration of HN2 (10nM) caused significant decreases in the IC₅₀ for A549 cell growthinhibition, as compared with treatment with each drug in the absence ofHN2 (see FIG. 3A), and these sensitizations were similar to the six-foldincrease in sensitivity when treating with 25 μM MK-571, a knownMRP1/mrp1 inhibitor. More specifically, treatment of cells with MRP1ligand etoposide inhibited growth of A549 cells (IC₅₀=1 μM).Co-treatment with 10 nM HN2, a concentration previously determined notto induce any growth inhibition in A549 cells, sensitized the cells toetoposide in a concentration-dependent manner (IC₅₀=75 nM). Because HN2does not cause any direct toxicity at this concentration, and inhibitionof MRP1 functional activity is also seen (see FIG. 1), thissensitization seen after co-treatment with HN2 and etoposide is likelydue to HN2 inhibiting MRP1 and the cells retaining etoposide, whichincreases its toxicity. It was further determined that the inhibition ofcalcein export by HN2 in A549 cells is reversible. Without being boundto any particular theory, these data suggest that HN2 is acting as areversible inhibitor of MRP1 by forming a conjugate to glutathione whichalso acts as a substrate/inhibitor for the transporter.

HN2 also enhanced the sensitivity of A549 cells to methotrexate andvincristine. Methotrexate inhibited growth (IC₅₀=230 nM), andco-treatment with 10 nM HN2 enhanced this cytotoxic effect (IC₅₀=48 nM).Vincristine also inhibited growth of A549 cells (IC₅₀=360 nM), andco-treatment with 10 nM HN2 also increased vincristine-induced growthinhibition (IC₅₀=45 nM). Methotrexate and vincristine are also MRP1substrates, and the concentrations of HN2 required to increase thesensitivity of A549 cells to methotrexate- and vincristine-inducedgrowth inhibition are also concentrations experimentally determined toinhibit MRP1-mediated efflux of calcein in these cells.

HN2 (10 nM) was also effective in sensitizing HEK cells overexpressingMRP1 to etoposide-induced growth inhibition (IC₅₀=960 nM for etoposidealone and 42 nM cells co-treated with 10 nM HN2). HEK control cells weremuch more sensitive to etoposide (IC₅₀=55 nM), and co-treatment with 3nM HN2 caused no change to etoposide-induced growth inhibition (IC₅₀=47nM). They are more sensitive to etoposide because they do not haveefflux transporters for the drug.

Accordingly, combination therapy with the nitrogen mustard HN2, as wellas other crosslinking agents, and MRP1 substrates with antitumoractivity can enhance the efficacy of many of these compounds when theyare used to treat tumors by formation of bifunctional adducts thatprevent the compounds from being effluxed from the cells. Because MRP1has been implicated in the export of anticancer agents from tumor cells,thereby limiting their efficacy, antagonism of this efflux pump is anattractive pathway to increase the sensitivity of antitumor agents. Asshown herein, the anticancer agent HN2 inhibits MRP1 functional activityat sublethal concentrations, potentially leading to a new breakthroughin understanding how to select the proper doses when using thisalkylating agent in combination with other MRP1 substrates, such asetoposide, methotrexate, and vincristine.

These experiments are indicative of the crosslinking agents andcompounds of the present invention also inhibiting or decreasingtransport of other drugs, efficacy of which is limited by MRP1-mediatedexport from cells, such as, but not limited to, protease inhibitorsamprenavir, ritonavir, and indinavir that are used to treat HIV/AIDS andantibiotics that act as substrates for MRP1, such as ciprofloxacin andgeprafloxacin.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1: Bifunctional Alkylating Agents Divinyl Sulfone,Diethyl Acetylenedicarboxylate and Nitrogen Mustard (HN2) Inhibit MRP1Efflux Drug Transporter Activity in A549 Tumor Cells

Bifunctional alkylating agents can form adducts with two glutathioneresidues. The inventors have discovered that these molecules have thecapacity to inhibit drug efflux transporters. This was demonstrated incells by showing that treatment with bifunctional alkylating agents canblock efflux of substrates of the efflux transporters. For thesestudies, A549 lung tumor cells were used. These cells were treated withincreasing concentrations of the bifunctional alkylating agents divinylsulfone, diethyl acetylenedicarboxylate or nitrogen mustard (HN2). Eachchemical can react intracellularly with glutathione to form abifunctional derivative. Calcein, a fluorescent MRP1 substrate, was usedto measure the effects of inhibitors on MRP1 activity as described byBircsak et al. (Curr. Protoc. Toxicol. 2013 57, Unit 23 6). Aftertreatment with a bifunctional alkylating agent, each compound was foundto inhibit calcein efflux from A549 cells in the nanomolar concentrationrange and in a concentration-dependent manner. See FIG. 1A showingdivinyl sulfone inhibits MRP1-mediated calcein efflux from A549 cells;FIG. 1B showing diethyl acetylenecarboxylate inhibits MRP1-mediatedcalcein efflux from A549 cells; and FIG. 1C showing nitrogen mustard(HN2) inhibits MRP1-mediated calcein efflux from A549 cells. The errorbars represent the means±SE, n=3.

Example 2: Effects of HN2 on the MRP1 Transporter

Bifunctional alkylating agents that have reacted with glutathione werealso shown to directly inhibit a cloned recombinant efflux transporter.In these studies, inside-out plasma membrane vesicles containingrecombinant MRP1 accumulate the fluorescent efflux transportersubstrate, glutathionyl bimane (bimane-GS). Treatment of the effluxtransporter with a glutathione bifunctional adduct blocks theaccumulation of the efflux transporter substrate. Bimane-GS was used tomonitor MRP1 functional activity in inverted membrane vesicles asdisclosed by Idasher et al. (Toxicology 2013, 306:108-113). Morespecifically, inside-out Sf9 insect cell plasma membrane vesiclesexpressing human MRP1 and those transfected with an empty vector(control) were purchased from Sigma-Aldrich (St. Louis, Mo.). To assessthe ability of HN2 to inhibit MRP1-mediated transport of bimane-GS, 20mg vesicles were incubated with 10 μM bimane-GS, 4 mM adenosinetriphosphate, 5 mM reduced glutathione, and 100 nM HN2 in reactionbuffer at 37° C. for 10 minutes according to the manufacturer'sprotocol. Vesicles were washed, vacuum filtered, and solubilized with50% methanol. Fluorescence was read at excitation wavelength 430 nm andemission wavelength 538 nm. Results are shown in FIG. 2. The control(open bar) shows low uptake of bimane-GS in vesicles that did notcontain the MRP1 efflux transporter. Vesicles containing the MRP1transporter (center filled in bar) readily take up bimane-GS. Uptake ofbimane-GS was blocked in vesicles containing the MRP1 transporter (rightfilled in bar) treated with the bifunctional alkylating agent, nitrogenmustard (HN2). The error bars represent the means±SE, n=3. The asterisksindicate that the bars are significantly different than vesiclescontaining MRP1 and not treated with HN2 (student t test).

Example 3: Ability of Nitrogen Mustard (HN2) to Enhance GrowthInhibition of A549 Tumor Cells by Cancer Chemotherapeutic Agents Knownto Efflux Via MRP1 Transporters

Many anticancer agents are limited in their effectiveness as they aresubstrates for the MRP1 drug efflux transporter and removed from cancercells. Findings disclosed herein that bifunctional alkylating agentsmodified with glutathione can effectively block the MRP1 effluxtransporter are indicative that they would have the capacity to enhancethe activity of chemotherapeutic agents that are MRP1 substrates byretaining them in the cancer cells. A549 lung tumor cells were used todemonstrate that a bifunctional alkylating agent (nitrogen mustard, HN2)can enhance cancer drug cytotoxicity as measured by growth inhibition.In these studies, A549 lung tumor cells were treated with non-cytotoxicconcentrations of HN2 (10 nM) and increasing concentrations of the drugsetoposide, methotrexate or vincristine, each of which is known to be asubstrate of MRP1. HN2, by reacting with intracellular glutathione (GSH)and forming bifunctional GSH adducts, inhibits efflux of the drugs andmarkedly enhances their sensitivity to etoposide, methotrexate orvincristine. Thus, lower concentrations of the drugs are able to inhibitcell growth in cells pretreated with HN2, which blocked the MRP1 effluxtransporter, when compared to cells not treated with HN2. In theseexperiments, growth inhibition was measured as described by Udasin etal. (Toxicological sciences: an official journal of the Society ofToxicology 2015), Mariano et al. (Biochemical pharmacology 2002,63(1):31-39), and Martey et al. (Biochem Pharmacol 2002,63(11):2001-2009). A549 cells were plated at low density (1.8-3.0×10⁴cells/well) in 24-well tissue culture dishes in growth medium andallowed to adhere overnight. The medium was then replaced with 0.35 mLof serum-free growth medium supplemented with 10 nM HN2 or control.After 1 hour, cells were then treated with increasing concentrations ofetoposide, methotrexate or vincristine. Thirty minutes later, the cellswere washed twice with HBSS and refed with fresh drug-free growthmedium. After an additional 72 hours, cells were removed from the disheswith trypsin and counted using a Z1 Coulter Particle Counter (BeckmanCoulter, Brea, Calif.). FIG. 3A shows that HN2 markedly enhanced growthinhibition of cells treated with etoposide. FIG. 3B shows that HN2markedly enhanced growth inhibition of cells treated with methotrexate.FIG. 3C shows that HN2 markedly enhanced growth inhibition of cellstreated with vincristine. The error bars represent the means±SE, n=3.

Example 4: Ability of Nitrogen Mustard (HN2) to EnhanceEtoposide-Induced Growth Inhibition in HEK Cells Expressing MRP1

Human embryonic kidney 293 (HEK) cells stably transfected to overexpressMRP1 (HEK MRP1) and those transfected with an empty pcDNA 3 vector (HEKcontrol) and thus do not express MRP1 (HEK) were used as described byRobey et al. (British journal of cancer 2003 89(10):1971-1978 andJournal of pharmacological and toxicological methods 201163(3):217-222). Cells were treated with non-cytotoxic concentrations ofHN2 (10 nM) and increasing concentrations of the drug, etoposide. HN2,by reacting with intracellular GSH and forming bifunctional adducts,inhibits MRP1-mediated efflux of etoposide and markedly increasessensitivity to this chemotherapeutic agent in HEK-MRP1 cells. Thus,lower concentrations of etoposide are able to inhibit cell growth. Incontrast, HN2 does not enhance sensitivity of HEK cells to etoposidesince they express little or no MRP1 transporter. In these experiments,growth inhibition was measured as described in Example 3. HEK andHEK-MRP1 cells were plated at low density (1.8-3.0×10⁴ cells/well) in24-well tissue culture dishes in growth medium and allowed to adhereovernight. The medium was then replaced with 0.35 mL of serum-freegrowth medium supplemented with 10 nM HN2 or control. After 1 hour,cells were then treated with increasing concentrations of etoposide.Thirty minutes later, the cell culture medium was supplemented withfetal bovine serum to a final concentration of 10%. After an additional72 hours, cells were removed from the dishes with trypsin and countedusing a Z1 Coulter Particle Counter (Beckman Coulter, Brea, Calif.).FIG. 4A shows HEK cells overexpressing MRP1 while FIG. 4B shows controlcells with little or no MRP1. Initial treatment with HN2 blocked theMRP1 efflux transporter in HEK cells overexpressing MRP1 (HEK MRP1) andincreased their sensitivity to further treatments with HN2. The errorbars represent the means±SE, n=3.

What is claimed is:
 1. A method for inhibiting or decreasing transportof a drug by a transporter of multidrug resistance-associated protein ina cell, said method comprising administering to the cell a compound ofFormula (VI)

wherein R₁ and R₂ are small peptides or modified peptides.
 2. The methodof claim 1 wherein R₁ or R₂ comprise glutathione.
 3. The method of claim1 wherein R₁ and R₂ comprise glutathione.
 4. The method of claim 1wherein efficacy of the drug is dependent on concentrations inside thecell.
 5. The method of claim 1 wherein the drug is selected from thegroup consisting of anti-inflammatory agents, neurological agents,thyroid agents, ocular agents, cancer chemotherapeutics, antibiotics,antimicrobials, antivirals and protease inhibitors to treat humanimmunodeficiency virus.
 6. The method of claim 1 wherein inhibiting ordecreasing transport of the drug by the transporter of multidrugresistance-associated protein potentiates action of the drug on thecell.
 7. The method of claim 1 wherein the cell is a cancer cell.
 8. Themethod of claim 1 wherein the cell is a microbe.
 9. The method of claim1 further comprising administration of a drug that is a substrate forthe transporter of multidrug resistance-associated protein.
 10. Apharmaceutical composition for inhibiting or decreasing transport of adrug by a transporter of multidrug resistance-associated protein in acell, said composition comprising a compound of Formula (VI)

wherein R₁ and R₂ are small peptides or modified peptides, and apharmaceutically acceptable carrier.
 11. The pharmaceutical compositionof claim 10 wherein R₁ or R₂ comprise glutathione.
 12. Thepharmaceutical composition of claim 10 wherein R₁ and R₂ compriseglutathione.
 13. The pharmaceutical composition of claim 10 furthercomprising a drug that is a substrate for the transporter of multidrugresistance-associated protein.
 14. The pharmaceutical composition ofclaim 10 wherein efficacy of the drug is dependent on concentrationsinside the cell.
 15. The pharmaceutical composition of claim 10 furthercomprising a drug selected from the group consisting ofanti-inflammatory agents, neurological agents, thyroid agents, ocularagents, cancer chemotherapeutics, antibiotics, antimicrobials,antivirals and protease inhibitors to treat human immunodeficiencyvirus.